Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome’s potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

. Based on our previous research, early rearing environments and both paternal and maternal factors influence offspring stress reactivity 11,43 , and we hypothesize that this reactivity may be mediated by alterations to the offspring gut microbiome via both heritable and environmental mechanisms.
In the current study, we aimed to: (1) measure the impact of paternal HFD feeding on offspring gut microbiome diversity and community composition; (2) explore the impact and overlap of early-life rearing environment and maternal care on the offspring gut microbiome; and (3) identify potential interactions between paternal diet and postnatal rearing conditions on offspring gut microbiome and their response to predator odor-induced stress. As described previously 11 , we fed F 0 males either control diet or HFD, bred them with females, and then measured maternal care of the offspring in the context of standard or semi-naturalistic housing (SNH) without the presence of the F 0 male, and we determined that the diversity and community structure of the F 1 offspring microbiome were significantly influenced by both paternal diet and maternal rearing environment. Together, these data support the hypothesis that paternal and maternal factors influence offspring gut microbiome and these changes influence behavior of the adult offspring.

Methods
Animals and breeding. All experimental procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals. Male (34) and female (30) Long-Evans hooded rats (64) (Charles River Canada, Sherbrooke, QC, Canada) were delivered to the vivarium at ~ 21-28 days old and maintained in the vivarium as same-sex pairs until adulthood (56 days old). All rats were maintained at 21 ± 2 °C under a 12 h reversed light cycle (lights off at 0930 h local time) in standard housing (SH), which consisted of polypropylene cages (47 cm length × 24 cm width × 20.5 cm height), wire lids, pine shavings for bedding (Hefler Forest Products, Inc., Sackville, NS, Canada), and a black polyvinyl chloride (PVC) tube (12 cm length × 9 cm diameter) for environmental enrichment, unless otherwise described (see below). For all rats, chow (Purina Lab Chow, Cat. No. 5001, Clarence Farm Services Ltd., Truro, NS, Canada) and water were supplied ad libitum. The research described here was conducted in compliance with the ARRIVE 2.0 Guidelines for Reporting Animal Research 66,67 . All efforts were made to limit the number of animals used and their suffering. Animals used in this study are identical to those used in a previous report; for a detailed description of animal procedures, see Korgan et al. 11 .
Paternal high-fat diet. A timeline of the experimental procedures is shown in Fig. 1A,B. F 0 males began either high-fat (60 kcal %; Product No. D12492) or protein/carbohydrate-matched control diet (10 kcal%; Product No. D12450J) feeding on PD35 and remained on this diet for 60 days (Research Diets, Inc., New Brunswick, NJ, USA). This diet is commonly used to induce DIO in rodents and mirrors the increased dietary fat and sugar content of a western diet 11,68,69 . Paired F 0 male cage-mates were randomly assigned either control diet (CD, n = 17) or high-fat diet (HFD, n = 17). On day 0, all F 0 males were supplied with standard rodent chow (Purina Lab Chow, Cat. No. 5001, Clarence Farm Services Ltd., Truro, NS, Canada) and water ad libitum until sacrifice. F 0 breeding. For breeding (Fig. 1A), one male and one naïve female (~ 56 days old), determined to be in estrus, were housed together for seven consecutive days; the male was then removed from the maternal cage. The day of birth was defined as postnatal day (PD) 0; pups remained with the dam until weaning at PD21 (Fig. 1B), upon which the F 1 offspring were re-housed in same-sex littermate pairs. Semi-naturalistic housing (SNH). F 0 females mated with CD and HFD F 0 males were observed daily for pups beginning at gestational day 20 (GD20), near the beginning of the dark cycle 11 . Once F 1 offspring arrived (PD0), the litter was counted, sexed, and weighed as quickly as possible to minimize disruption. F 0 females and F 1 offspring randomly designated for the SNH condition (n = 9 dams, n = 40 offspring) were transferred to SNH cages on PD0. Dams and pups in the SH condition (n = 10 dams, n = 38 offspring) were placed in clean SH (Fig. 1C). All F 0 dams and F 1 pups were left undisturbed until the pups were weaned at PD21. The SNH (Fig. 1C) consisted of two sections: an upper section (50.5 cm width × 50.5 cm length × 33.5 cm height) containing chow food and water ad libitum and a lower section (50.5 cm width × 50.5 cm length × 14 cm height) filled with pine shavings and a polyvinyl chloride (PVC) tube.
Maternal care observations. Maternal behaviors of F 0 females were scored daily in real-time for 72 min at 0800 h, 1100 h, 1300 h, 1500 h, and 2130 h from PD1 to PD7 (Fig. 1B). During each observation period, the frequencies of the behaviors were determined using an instantaneous sampling method, recording the behavior occurring every 3 min 11,47 . For the current study, we assessed arched-back-nursing (ABN), with licking and grooming (LG) of pups. ABN consisted of graded degrees of arching, levels 2-4, based on the presence of kyphosis (bending of the knees and back arching) while nursing F 1 offspring. The sum of high-quality maternal behaviors towards the offspring (i.e., the total number of occurrences of LG, ABN2, and ABN3; representing moderate and moderate/high degrees of kyphosis) were divided into three groups to form equal sized "high", "medium", and "low" maternal care groups for further analyses.
Offspring groups. The following four groups of F 1 male and F 1 female offspring were utilized: (1) CD-SH-F 0 male exposed to control diet and F 0 female housed in standard housing (n = 14 males, 11 females); (2) CD-SNH-F 0 male exposed to control diet and F 0 female housed in semi-naturalistic housing (n = 8 males, n = 10 females); (3) HFD-SH-F 0 male exposed to high-fat diet and F 0 female housed in standard housing (n = 6 males, n = 7 females); and (4) HFD-SNH-F 0 male exposed to high-fat diet and F 0 female housed in semi-naturalistic Stomach microbiome sample collection. Stomach microbiome sample collection was conducted in a sterile BSL2 cabinet using sterile technique. Stomachs, individually wrapped in aluminum foil, were removed from the − 80 °C freezer, thawed on wet ice, and then dissected along the lesser curvature with a sterile scalpel  Males were fed either control diet (CD) or high-fat diet (HFD) for 60 days prior to a partner preference test (PPT) using sexually receptive, naïve females. Within 12 h of the PPT, males were bred with different receptive naïve females. Following mating, males were removed and F 0 females were left undisturbed until postnatal day (PD) 0. After mating, F 0 males were maintained on standard chow for 21 days before a second PPT. One week after the second PPT, F 0 male anxiety-like defensive behavioral responses were assessed in the open-field test (OFT) and the elevated plus-maze (EPM). Following behavioral testing, F 0 males were immediately sacrificed; stomachs were collected, frozen and stored at − 80 °C for microbiome processing. (B) Timeline of treatment procedures for F 0 females and the F 1 offspring. After birth (postnatal day 0, PD0), offspring were counted, sexed, and weighed before being transferred to either fresh standard housing (SH) or semi-naturalistic housing (SNH), with biological mothers, until weaning. Maternal behavior (Maternal care) was scored for observational periods of 72 min, five times per day for seven consecutive days. At PD21, all offspring were weighed, weaned and placed in SH with a same-sex littermate. Play behavior was recorded in the home cage from PD24-29, followed by testing in the OFT and EPM on PD32 and 35, respectively. Predator odor exposure (OE) testing took place on PD42 with male and female offspring exposed to either a control odor (CO) or predator odor (PO) for 30 min, followed immediately by sacrifice. Fecal pellets and stomachs were collected, frozen and stored at − 80 °C for microbiome processing. Sample sizes are provided in the figure for both F 0 and F 1 groups. (C) Schematics of the SH and SNH. The SNH consisted of a lower burrow compartment contained within a drawer that could be moved out to facilitate cage cleaning and an upper section that contained food and water; the two sections were connected by a hole (visible in the upper section Stomach and fecal microbiome analysis. Demultiplexed, quality-filtered reads were analyzed using the QIIME 2 2019.4 workflow with default parameters 72 , generating error-corrected amplicon sequence variants (ASVs) using the DADA2 (ver. 1.10.0) denoising and dereplication workflow 73 . Stomach samples averaged 6758.7 ± 436.8 (mean ± standard error of the mean) sequences per sample, with the minimum number of sequences required to retain a sample in the study set at 2200 for F 0 sires and 1850 reads for F 1 offspring to maximize the number of samples retained. Except where noted, the raw OTU table was rarefied to correct for differential sequencing effort and resultant library size artifacts. Although rarefaction is a conservative approach that limits power for discovery of differences, it more clearly clusters samples according to biological origin in presence/absence ordinations (i.e., unweighted UniFrac) than other normalization techniques and is an essential prerequisite to conducting α-and β-diversity analyses 74 . Seven samples were excluded due to insufficient sequence reads (less than 90) from the following animal subjects: Rat IDs 9, 18, 19, 21, 26, 28, and 36. Reads from the 34 remaining samples were clustered into sub-OTUs against the Greengenes 13/8 reference database as above. Representative sequences were then aligned with MAFFT and a phylogenetic tree was constructed with FastTree 2 for phylogenetic diversity calculations. In summary, microbial data from 34 of the original 41 stomach samples were analyzed for a variety of alpha diversity metrics (for details, see below).
Fecal samples averaged 9381.3 ± 619.7 (mean ± standard error of the mean) sequences per sample, with the minimum number of sequences required to retain a sample in the study set at 3136 reads to maximize the number of samples retained. Four samples were excluded due to insufficient sequence reads (less than 2000) from the following animal subjects: Rat IDs 43, 59, 67, and 77. Reads from the 44 remaining fecal samples were clustered into sub-OTUs against the Greengenes 13/8 reference database 75,76 . Representative sequences were then aligned with MAFFT (ver.7.0) 77 , and a phylogenetic tree was constructed with FastTree 2 (ver. 2.0) 78 for phylogenetic diversity calculations. In summary, microbial data from 44 of the original 48 fecal samples were analyzed for a variety of alpha diversity and beta diversity metrics (for details, see below). Additionally, we utilized a supervised machine-learning algorithm to predictively classify individual fecal microbiome samples of male and female offspring as belonging to either F 0 paternal CD or HFD, F 0 maternal/ early-life F 1 offspring housing SH or SNH, or F 0 maternal care quality; high, medium, or low. Using a nested cross-validated (k = fivefold) strategy, the receiver-operating characteristic (ROC) area-under-the-curve (AUC) values for the ASV-based models that were used to calculate feature importances varied between 0.77 and 0.95.

Statistical analyses.
Statistical analysis in QIIME2 of each alpha-diversity metric made available in the command-line interface was conducted on both the stomach and fecal microbiome sequencing data (Chao1 richness estimator, Shannon's entropy Hʹ, Menhinick's richness index, and Simpson's evenness index. Briefly, once each alpha-diversity metric above was calculated for stomach microbiome samples from F 0 sires and F 1 offspring and fecal samples from F 1 offspring, statistical analysis was conducted using Kruskal-Wallis tests and post hoc pairwise Mann-Whitney U tests with adjustment for multiple comparisons on all categorical variables in the metadata. For the stomach sample metadata from the paternal generation/F 0 sires, these variables included: final weight at necropsy, dietary condition (CD or HFD), weight gain from pre-to post-diet manipulation, testes weight, abdominal fat pad weight, gonadal fat weight, brain weight, and percentages of each weight taken relative to the final weight at necropsy.
For both the stomach and fecal sample metadata relevant to only the F 1 offspring, we conducted statistical analysis as described above against the following variables: sex (male or female), filial generation (F 0 or F 1 ), paternal dietary condition (CD or HFD), F 0 maternal/early-life F 1 offspring housing (SH or SNH), F 1 stress exposure condition (CO or PO), F 0 maternal care quality (licking/grooming and arched-back nursing), Crf hnRNA expression, and expression of H3K9ac at the Crf promoter. In the fecal sample metadata, F 1 offspring-specific , grooming frequency in the OF, and rearing frequency in the OF. F 0 sire and F 1 offspring behavioral metadata for the EPM and OF as just described were also included in the alpha-diversity analysis of the stomach microbiome sequencing data. Only the significant drivers of variation in the F 1 offspring are reported in the results section and can be found in Supplementary Tables 1-3. Alpha-diversity analyses of Crf hnRNA expression and H3K9ac at the Crf promoter, and behavioral metadata for the EPM and OF were found to be not significant. Raw data points for each of the metadata columns described can be found in Supplementary File 2 and 3. Additional statistical analysis of core and commonly reported alpha-diversity metrics (number of distinct features, Shannon's entropy, and Faith's phylogenetic diversity) using 3-way linear mixed effects models and post hoc pairwise t-tests without adjustment for multiple testing were also conducted on the fecal microbiome sequencing data. Following rarefaction of the ASV table generated by 16S rRNA gene sequencing of all fecal samples briefly described above, we conducted a linear mixed effects model-based 3-way analysis of F 0 paternal diet (CD or HFD), F 0 maternal/early-life F 1 offspring housing (SH or SNH), and F 1 stress exposure conditions (CO or PO). For beta diversity distances, such as weighted and unweighted UniFrac, similarly structured 3-way permutational analyses of variance of F 0 paternal diet, F 0 maternal/early-life F 1 offspring housing, and F 1 stress exposure conditions were conducted using the R package adonis (999 Monte Carlo permutations).

Results
Paternal HFD influences offspring weight and anxiety-like behavior. We showed that male and female F 1 offspring of F 0 HFD sires were significantly heavier on PD42 compared to offspring of F 0 CD sires (one-way analysis of variance, F (3, 44) = 17.91, p < 0.0001; Fig. 2A). We also showed that F 1 male offspring weighed more than F 1 female offspring (two-way analysis of variance, F (1,44) , p = 0.0004) and that male and female F 1 offspring of F 0 HFD sires were significantly heavier on PD42 compared to male and female offspring, respectively, of F 0 CD sires (two-way analysis of variance, F (1, 44) = 17.91, p < 0.0001; Fig. 2A); this is despite the fact that F 1 offspring were fed control chow from weaning through PD42. These data are consistent with previous findings indicating that F 0 paternal HFD feeding negatively impacted mating success and subsequent F 0 maternal care of F 1 offspring 11,43 which has also been shown to affect F 1 offspring bodyweight 51 . Additionally, we examined whether correlations exist between maternal care, F 0 paternal HFD treatment, offspring anxiety-related defensive behavioral responses, and F 1 offspring weight (PD42). Pearson's correlation analysis demonstrated that increased frequency of F 0 maternal behaviors scored pre-weaning (PD1-7) were inversely associated with F 1 offspring weights taken at PD42 (r = − 0.614, p < 0.0001; Fig. 2B). Further, body weight was negatively correlated with percentage of time in the open arms of the elevated plus-maze, (r = − 0.263, p = 0.0003; Fig. 2C). Statistical significance was evaluated using a pairwise t-test within F 1 sexes. **p < 0.01, ****p < 0.0001. CD control diet, HFD high-fat diet. www.nature.com/scientificreports/ Together, these results highlight the link between F 0 maternal care, F 0 paternal diet, and anxiety-like behavior in F 1 offspring.

Maternal housing and anxiety-like behaviors in offspring impact the gastric microbiota.
Although the stomach microbiome is known to have lower diversity than the intestinal microbiome, we selected the stomach microbiome as representative of the microbial continuity of the aerodigestive tract 79 , which is thought to play an important, albeit understudied, role in modulation of physiology and behavior. We conducted Kruskal-Wallis tests and post hoc pairwise Mann-Whitney U tests with adjustment for multiple testing on all categorical variables in the metadata describing each sample submitted for 16S rRNA gene sequencing of the stomach microbiomes. Samples were collected from F 0 CD and HFD sires and F 1 offspring from F 0 paternal diet (CD or HFD), F 0 maternal/ early-life F 1 offspring housing (SH or SNH), and F 1 stress exposure conditions (CO or PO). A full summary of significant findings for F 0 CD and HFD sires is in Supplemental Table 1; significant findings for F 1 offspring are summarized in Supplemental Table 2.
Briefly, the most consistent effects that we observed among F 0 sires were differences in multiple alpha-diversity metrics of evenness and richness in association with diet ( Paternal diet, maternal housing, and stress exposure interactions impact gut microbial phylogenetic diversity and richness. Because previous literature indicates that changes in stress responsivity and body weight may be associated with changes in the gut microbiome, we conducted three-way linear mixed-effects models to evaluate the potential main and interactive effects of F 0 paternal diet (CD or HFD), F 0 maternal/early-life F 1 offspring housing (SH or SNH), and F 1 stress exposure conditions (CO or PO) on microbial alpha-diversity. As described in "Molecular processing of microbiome samples" section, the 16S rRNA gene V4 amplicon data were first subsampled at a rarefaction depth of 3136 reads per fecal sample collected from 44 F 1 offspring in the test arena at the conclusion of the odor exposure (OE) test, immediately prior to sacrifice.
Analysis of Faith's phylogenetic diversity revealed interaction effects of paternal diet × maternal housing × stress exposure (F (1,36) = 6.2, p = 0.018; Fig. 3A Fig. 3C). However, no main effects of F 0 paternal diet, F 0 maternal/ early-life F 1 offspring housing, or F 1 stress exposure were found in any of the linear mixed-effects models described above.
Paternal diet with maternal housing or stress exposure impacts global composition of the gut microbiota. To assess effects of paternal diet, maternal housing conditions, and stress exposure on the community composition of fecal microbiomes, we conducted three-way permutational analysis of variance (999 permutations) using the R package adonis on the unweighted and weighted UniFrac distance matrices generated from the rarefied feature table. Analysis of weighted UniFrac in the between-subjects comparisons showed no significant differences in microbial beta-diversity (data not shown). However, we demonstrated the presence of an interaction effect of paternal diet × maternal housing condition (F (1,36) = 1.59, r 2 = 0.036, p = 0.044; Fig. 4A,B) and paternal diet × stress exposure (F (1,36) = 1.52, r 2 = 0.034, p = 0.050; Fig. 4A,C) in the unweighted UniFrac distance matrix data.
HFD offspring have elevated Firmicutes to Bacteroidetes ratios compared to CD offspring. The relative abundances of all phyla identified in the rarefied feature table of offspring fecal microbiomes are shown in Fig. 5A. From the stacked barplot, over 90% of the gut microbiota of all 44 F 1 offspring fecal samples were members of the phyla Firmicutes, Bacteroidetes, and Verrucomicrobia. We generated a principal coordinates analysis (PCoA) plot of the unweighted unique fraction metric (UniFrac) distances with compositional biplot vectors representing the contributions of these 3 phyla to beta-diversity clustering patterns in the offspring of CD and HFD sires (Figs. 4A, 5B). Here, we demonstrated that high levels of Firmicutes corresponded to fecal samples belonging to F 1 offspring of HFD sires (Fig. 5B). Analyses of the fecal samples of F 1 offspring with logtransformed relative abundance ratios of Firmicutes to Bacteroidetes (log 10 F:B ratio) > 0 demonstrated that 16 samples belonged to progeny of F 0 HFD sires and an additional 11 samples with (log 10 F:B ratios) > 0 belonging to progeny of F 0 CD sires had greater than 10% prevalence of Verrucomicrobia (Fig. 5A,C). Fisher's Exact probability test identified a significant difference between the relative abundance ratios of Firmicutes to Bacteroidetes (log 10 F:B ratio) > 0 for CD and HFD F 1 offspring (OR 4.49, p = 0.033), with a greater number of F 0 HFD sires having Firmicutes to Bacteroidetes (log 10 F:B ratio) > 0.
Using microbial features in the offspring fecal microbiota to accurately predict parental conditions. A nested cross-validation random forest approach (5 k-mers) was used to evaluate sample classification by F 0 condition (paternal diet, Fig. 6A-C; maternal housing condition, Fig. 6D   Unbiased classification of samples based on microbial composition extended our understanding of how the fecal microbiota of F 1 rat offspring in this study were influenced by F 0 conditions beyond core diversity analyses and relied on predictive accuracy scores and machine learning model performance indicators in the receiver operating characteristic (ROC) scores (Fig. 6A,D,G). Briefly, macro-and micro-average values for the prediction of samples to F 1 offspring of CD or HFD sires based on features of the fecal microbiome were greater than chance alone (AUC = 0.95 and 0.95, respectively; Fig. 6A) and 95% accurate (Fig. 6B). The feature importance plot in Fig. 6C shows that the presence or absence of Ruminococcus and Lactobacillus spp. together drove the classification of samples into paternal CD or HFD progeny. Additional features included Blautia sp., Ruminococcus flavefaciens, Bifidobacterium pseudolongum, and unknown members of the Bacteroidetes class S24-7, family Rikenellaceae, order Clostridiales, and order Mollicutes RF39 (Fig. 6C).
Macro-and micro-average values for the prediction of samples to F 1 offspring reared with dams in SH or SNH environments based on features of the fecal microbiome were also greater than chance alone (AUC = 0.85 and 0.85, respectively; Fig. 6D) and 84% accurate (Fig. 6E). The feature importance plot in Fig. 6F demonstrates that there exists some overlap in features used in the classification of samples by paternal diet and maternal housing condition, including Blautia sp. and an unknown member of the Bacteroidetes class S24-7 (Fig. 6C,F). Unique features that drove the classification of SH and SNH conditions included unknown members of the family Ruminococcaceae and Clostridiaceae, Allobaculum sp., Turicibacter sp., Prevotella sp., Akkermansia muciniphila, and multiple features in the genus Lactobacillus (Fig. 6F).  0.81, 0.77, and 0.84, respectively; Fig. 6G). Predictive accuracies for each classification level shown in Fig. 6G and the confusion matrix in Fig. 6H Fig. 6G). The feature importance plot in Fig. 6I demonstrates that here, too, there is substantial overlap in features used in the classification of samples by maternal care and housing condition, including Blautia sp., Turicibacter sp., and an unknown member of the family Ruminococcaceae. As in prior analyses, there were also some unique features that drove the classification of samples into recipients of high, medium, or low maternal care such as Lactobacillus sp. and Clostridium sp. (Fig. 6I). Additional unique features that were not well characterized at the species level included unknown members of the order Clostridiales, family Peptostreptococcaceae, and subfamily Clostridiaceae 02d06 (Fig. 6I).
Linear mixed-effects model analysis of paternal diet and maternal housing condition using ANCOM-II identified one significant feature above the coefficient of concordance threshold of 0.9 in the F 1 fecal microbiome data (Fig. 7A). The observed relative abundance of Bifidobacterium pseudolongum (W-statistic = 40) in the offspring of HFD sires was significantly higher compared to offspring of CD sires in all subgroups irrespective of maternal housing condition or odor exposure prior to sacrifice (Fig. 7B).

Discussion
In the current study, we have identified novel interactions between preconception paternal diet, postnatal rearing conditions, maternal care, and predator odor-induced stress that impact the offspring microbiome. Specifically, we have identified correlations between paternal diet factors and maternal investment which influence offspring weight and anxiety-like behavior, microbiome alpha-and beta-diversity, and Firmicutes to Bacteroidetes ratios. Further, we identified that compositional dynamics between members of the phyla Firmicutes and Bacteroidetes principally drove these treatment group differences, while also identifying Bifidobacterium pseudolongum, a member of the phylum Actinobacteria, as an important individual species that was differentially-abundant based on paternal diet condition.
Correlational analyses suggest that there was an interaction between paternal diet and maternal investment, and these interactions influence offspring weight throughout life and adult anxiety-like behavior. Maternal investment is dictated by many factors, including the quality of the mate and previous and current environmental conditions 20,21,81 . Previously, we identified decreases in the quality of maternal care in offspring from stressed or HFD-fed F 0 males and that deviations in maternal investments have long-term impacts on offspring growth and behavior 11,43 . Conversely, offspring of males in an enrichened environment received higher quality maternal care and had improved learning and memory 20,82 . We identified significant interactions in F 1 male and female offspring weight; predictably, (1) males weighed more than females, (2) offspring of F 0 sires fed HFD weighed more than offspring of F 0 sires fed CD. This weight increase was negatively correlated with the quality of maternal care and associated with increased anxiety-like behavior; these findings suggest there is an interaction between paternal diet, early-life experience, maternal investment, and adult anxiety-like behavior. While previous studies have suggested that high quality environment can influence maternal care and offspring development 51,83 , acute stress www.nature.com/scientificreports/ exposure in peri-adolescent rats, relative to unstressed controls, has not been extensively studied to identify differences in behavior, gene expression, epigenetic regulation, and gut microbiota composition. To probe the potential interactions of paternal diet, maternal care/offspring weaning environment, and acute stress exposure on the gut microbiome, we analyzed fecal samples from F 1 offspring following predator odor exposure (or control). Next, we demonstrated that there are interactive effects of paternal diet and maternal investment on offspring gut microbial alpha-diversity. Previous studies of paternal and maternal HFD feeding have detailed robust differences in alpha-diversity in F 1 offspring 60,84 . While we do not replicate the diet-induced differences in diversity, our model excluded maternal HFD-feeding and eliminated HFD metabolites in breast milk, unlike other studies. Other studies with controlled paternal-only feeding have shown similarly limited effects of paternal prebiotic 85 and sucrose diets 86 on alpha diversity in F 1 offspring microbiomes. Interestingly, we identified several significant alterations in alpha diversity based on offspring odor exposure. The acute nature (30 min) of the PO exposure, immediately prior to sample collections, suggests the possibility of stress-induced catecholaminergic effects on microbiome diversity 87,88 that are dependent on offspring rearing environment.
We found that these interaction effects were also observed in compositional analyses of microbial betadiversity, such that paternal high-fat diet drives the presence and absence of keystone species in offspring. Again, like previous studies of paternal and/or maternal HFD feeding 84,89 , we show a more subtle effect of paternal HFD on offspring gut microbiomes, similar to paternal prebiotic 85 and sucrose diets 86 . We identified a profound influence of rearing environment, supporting previous evidence that identified early life care and stress as key drivers of offspring microbiome beta-diversity 57,90 . We also identified beta diversity differences based on PO exposure, again suggesting an impact of catecholaminergic signaling on acute microbiome changes 87,88 .
We demonstrated that paternal HFD exposure increases the ratio of Firmicutes to Bacteroidetes in offspring, agreeing with previous epigenetic studies 60 . This subtle shift in the prevalence of Firmicutes also reflects the effects seen in obesity and prenatal stress 90,91 and further supports the role of paternal nutrition in shaping offspring growth and behavior 7,10,11,22,25,85,92,93 . The role of other predominant phyla in the rat gut microbiota, namely Verrucomicrobia and Actinobacteria, are not as clearly elucidated in the context of diet in combination with generational-induced changes. However, Verrucomicrobia (and certain probiotic members of that phylum, i.e., Akkermansia muciniphila), a species with anti-inflammatory and immunoregulatory properties [92][93][94][95][96][97] , emerged as one of the top three drivers of effects of paternal diet on F 1 offspring community composition (Fig. 5B). Additionally, A. muciniphila has been shown to increase glucose homeostasis in diet-induced obesity [96][97][98][99][100] . Predictive random forest modeling demonstrates a clear delineation between members of Firmicutes, such as Ruminococcus sp. and Lactobacillus sp., and members of the Actinobacterium phylum, such as Bifidobacterium pseudolongum, in determining the classification of offspring gut microbiomes. Surprisingly, paternal HFD and associated gut microbial changes had the strongest predictive accuracy in determining offspring classification of the experimental factors analyzed despite more temporally proximal events taking place during the offspring's lifespan (i.e., maternal care and odor exposure). Maternal investment indicators such as nursing, licking, and grooming early in the offspring life period also are influenced by paternal high fat diet 11 and thus have strong predictive accuracy.
Bifidobacterium pseudolongum was highly expressed in the offspring of HFD sires, specifically those raised in SNH conditions. This species has recently been shown to reduce body weight, visceral fat, and reverse HFDinduced increases in Firmicutes to Bacteroidetes ratios 101 . We hypothesize that Bifidobacterium pseudolongum potentially acts as a compensatory response within the gut microbiota to elevated levels of Firmicutes. Bifidobacterium pseudolongum is also associated with decreases in Akkermansia muciniphila, replicated in offspring reared in SNH, and increased colonic mucus layer thickness 102 . However, the specific subspecies and strain identified here would need to be known for potential probiotic treatment, as Bifidobacterium pseudolongum has many strains with varied metabolic effects 103 . Future work will also be required to disentangle the influence that paternal diet, maternal care and rearing environments have on differential expression of these buffering microbiome species.
Recent research has identified several potential routes for non-genetic inheritance of paternal experience, including the seminal microbiome, epigenetic alterations to sperm, paternal inheritance of mitochondrial DNA, and maternal investment. For example, diet-induced alterations to paternal microbiome may be transferred to offspring via the seminal fluid microbiome 28,104 . Others have identified specific alterations in sperm epigenetic mechanisms, including small noncoding RNAs (sncRNAs), tRNAs 22,24,29 , and microRNAs 25,26,105 ; these mechanisms alter gene expression of developing embyos 106 and likely contribute to metabolic insults in offspring 10,25,86 . However, the role of maternal investment should not be overlooked. We have previously demonstrated that female rats have a preference for lean males when given the option between a lean vs. DIO male 11 and this is reflected in quality of maternal care. Others have identified increased maternal behavior based on the female's perceived quality of a mate [19][20][21] . However, the total impact of transgenerational insults has not been explored in the context of mate preference. The influence of maternal preference can be reduced by utilizing in vitro fertilization 9,24,107 or cross-fostering techniques [105][106][107][108][109] , though both of these techniques can alter development 110,111 . Maternal investment can also be manipulated by the quality of the environment, as an enriched or naturalistic rearing environment will promote a higher proportion of high-quality vs. low-quality maternal care 11,43,51,83 . Limitations. While the statistical analyses and data reported here are robust and replicable, we acknowledge that there are some limitations to this study that prevent us from concluding the underlying functional mechanism(s) by which paternal high fat diet, maternal housing, and acute stress (predator odor exposure) influence the gut microbiota throughout the offspring lifespan. Fecal and stomach sampling took place at a single discrete timepoint in F 1 offspring peri-adolescence 112 ; future studies using longitudinal sampling would permit us to determine if factors such as maternal care or rearing condition immediately impact the offspring microbiome or if these changes are the result of gradual interactions between maternal care and rearing condition. www.nature.com/scientificreports/ Future work should also consider longer-lasting and aging-related changes in offspring as they progress from peri-adolescence to adulthood while considering the possibility that these changes may expand to future generations as well. Moving forward, careful design and implementation of experiments investigating these complex and interacting factors will be required to fully elucidate the influence of paternal experience on maternal investment and rearing environment on offspring metabolism and behavior.

Conclusions
Preconception and early-life factors have a significant influence on the development and adult behavior of many organisms, including mammals. Here, we show that preconception paternal HFD feeding, early-life rearing environment, and maternal care influence offspring weight, behavior and the diversity of their microbiome. Specifically, we have identified that paternal HFD positively associates with offspring weight and anxiety-like behaviors in peri-adolescence, and maternal investment inversely associates with offspring weight. We show that predator odor exposure is an acute stressor that impacts offspring gut microbiota as assessed by richness and evenness indices of alpha-diversity as well as global compositional changes in beta-diversity measurements. These data suggest that parental conditions such as paternal HFD and maternal care, together with acute stress exposures during early life, could potentially impact the gut microbiota through adulthood. Future studies are required to determine the germline mechanisms driving these generational, and potential transgenerational, effects. However, the role of maternal investment should not be overlooked 21 when determining priming of offspring development and behavior by paternal experience. Overall, these data support the hypothesis that both paternal diet and maternal care have profound influence on offspring microbiota diversity and community composition, and that these changes influence the behavior of peri-adolescent offspring.

Data availability
Mapping files and raw short-read DNA sequences are publicly available on the microbiome study management platform Qiita (http:// qiita. ucsd. edu/ study/ descr iption/ 1634) and the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI, European Nucleotide Accession No. ERP015380). Other data from the study are available from the corresponding author on reasonable request.